Summary Although it has been known for decades that the ST6Gal-I sialyltransferase is upregulated in cancer, there is still a striking dearth of information regarding the functional role of ST6Gal-I in regulating tumor cell behavior. Our group has been at the forefront of characterizing the specific ST6Gal-I receptor substrates that reprogram tumor cell signaling to impart a stem-like, metastatic phenotype. These studies implicate ST6Gal-I as a master regulatory molecule that controls the function (via sialylation) of key receptors including TNFR1, Fas, EGFR and ?1 integrin. Together this receptor cohort directs the activation of signaling cascades that promote hallmark cancer stem cell (CSC) characteristics such as invasiveness and apoptosis-resistance. In recent unpublished work, we have definitively confirmed a tumor-driver role for ST6Gal-I in pancreatic ductal adenocarcinoma (PDAC). We generated a new genetically engineered mouse with ST6Gal-I overexpression in the pancreas, and crossed this line to the ?KC? pancreatic cancer model, which harbors oncogenic K-ras expression in the pancreas. Compared to KC mice, KC mice with ST6Gal-I overexpression have greatly accelerated PDAC development, metastasis, and mortality. We hypothesize that ST6Gal-I activity contributes to two critical steps in pathogenesis: (1) acinar-to-ductal metaplasia (ADM), an early event in PDAC initiation (Aim 1), and (2) PDAC metastasis (Aim 2), which is the chief cause of patient mortality. In Aim 1 we will elucidate molecular mechanisms by which ST6Gal-I promotes ADM, focusing on the hypothesis that ST6Gal-I activates NF-?B signaling to induce Sox9 expression. A robust literature has established that upregulation of Sox9 plays an essential role in ADM, however the finding that ST6Gal-I-mediated sialylation controls Sox9 expression points to a transformative role for tumor glycosylation in PDAC initiation. The importance of a ST6Gal-I/NF-?B/Sox9 signaling axis in ADM will be evaluated in: (i) the canonical 266-6 ADM cell model; (ii) epithelial organoid lines derived from our various mouse models; and (iii) the in vivo ADM models, ductal ligation and cerulein injection. In Aim 2 we will interrogate the sialylation-dependent signaling mechanisms that reprogram tumor cells into CSC-like cells with metastatic capability. Our central premise is that ST6Gal-I- mediated sialylation of EGFR, TNFR1/Fas, and ?1 integrin acts as a molecular switch to alter signaling nodes that confer stem-like properties. Furthermore, we will test the game-changing hypothesis that exosomal transfer of active ST6Gal-I to recipient cells can reprogram these cells to acquire metastatic properties. To verify that ST6Gal-I plays a causal role in metastasis we will use bioluminescence imaging to track the metastatic dissemination of orthotopically implanted PDAC patient organoids, as well as Suit2 cells and their isogenic metastatic clones. In the aggregate, these studies are expected to reveal an unprecedented role for tumor cell sialylation in driving PDAC progression.